ABSTRACT
Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.
Subject(s)
GTP Phosphohydrolases , Membrane Fusion , Mitochondria , Mitochondrial Membranes , Humans , Biocatalysis , Cardiolipins/chemistry , Cardiolipins/metabolism , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/metabolism , Mutation , Protein Domains , Protein Multimerization , Mitochondrial DynamicsABSTRACT
Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
Subject(s)
Cryoelectron Microscopy , Electron Transport Complex III , Electron Transport Complex II , Electron Transport Complex IV , Electron Transport Complex I , Mitochondria , Mitochondrial Membranes , Electron Transport , Electron Transport Complex III/chemistry , Electron Transport Complex III/metabolism , Electron Transport Complex III/ultrastructure , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Electron Transport Complex IV/ultrastructure , Mitochondria/chemistry , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/ultrastructure , Electron Transport Complex II/chemistry , Electron Transport Complex II/metabolism , Electron Transport Complex II/ultrastructure , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Electron Transport Complex I/ultrastructure , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Molecular Dynamics Simulation , Binding Sites , Evolution, MolecularABSTRACT
Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)-based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.
Subject(s)
Electron Transport Complex III/metabolism , Electron Transport Complex II/metabolism , Electron Transport Complex IV/metabolism , Electron Transport Complex I/metabolism , Mitochondria/enzymology , Myocardium/enzymology , Proteome/metabolism , Animals , Binding Sites , Brain/enzymology , Citric Acid Cycle/genetics , Electron Transport Complex I/chemistry , Electron Transport Complex I/genetics , Electron Transport Complex II/chemistry , Electron Transport Complex II/genetics , Electron Transport Complex III/chemistry , Electron Transport Complex III/genetics , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Gene Expression , Half-Life , Lipid Metabolism/genetics , Mice , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Models, Molecular , Organ Specificity , Oxidative Phosphorylation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Stability , Proteome/chemistry , Proteome/geneticsABSTRACT
Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and ß (IKKα/ß) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK-/- cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.
Subject(s)
Brain Neoplasms/enzymology , Energy Metabolism , Glioblastoma/enzymology , Mitochondria/enzymology , Protein Serine-Threonine Kinases/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Dynamins/genetics , Dynamins/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Dynamics , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/pathology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Tumor Microenvironment , NF-kappaB-Inducing KinaseABSTRACT
Mitochondria are essential organelles that execute and coordinate various metabolic processes in the cell. Mitochondrial dysfunction severely affects cell fitness and contributes to disease. Proper organellar function depends on the biogenesis and maintenance of mitochondria and its >1,000 proteins. As a result, the cell has evolved mechanisms to coordinate protein and organellar quality control, such as the turnover of proteins via mitochondria-associated degradation, the ubiquitin-proteasome system, and mitoproteases, as well as the elimination of mitochondria through mitophagy. Specific quality control mechanisms are engaged depending upon the nature and severity of mitochondrial dysfunction, which can also feed back to elicit transcriptional or proteomic remodeling by the cell. Here, we will discuss the current understanding of how these different quality control mechanisms are integrated and overlap to maintain protein and organellar quality and how they may be relevant for cellular and organismal health.
Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitochondria/enzymology , Mitochondria/genetics , Mitochondria/ultrastructure , Mitochondrial Dynamics , Mitochondrial Membranes/enzymology , Mitophagy , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Transport , Transcription, Genetic , Ubiquitin/metabolismABSTRACT
Manganese porphyrins are well-known protectors against the deleterious effects of pro-oxidant species such as superoxide ions and hydrogen peroxide. The present study investigated the antioxidant cytochrome c-like activities of Mn(III)TMPyP [meso-tetrakis (4-N-methyl pyridinium) porphyrin] against superoxide ion and hydrogen peroxide that remained unexplored for this porphyrin. The association of TMPyP with a model of the inner mitochondrial membrane, cardiolipin (CL)-containing liposomes, shifted +30 mV vs. NHE (normal hydrogen electrode) redox potential of the Mn(II)/Mn(III) redox couple. In CL-containing liposomes, Mn(III)TMPyP was reduced by superoxide ions and recycled by Fe(III)cytochrome c to the oxidized form. Similarly, isolated rat liver mitoplasts added to a sample of Mn(II)TMPyP promoted immediate porphyrin reoxidation by electron transfer to the respiratory chain. These results show that Mn(III)TMPyP can act as an additional pool of Fe(III)cytochrome c capable of transferring electrons that escape from the IV complex back into the respiratory chain. Unlike Fe(II)cytochrome c, Mn(II)TMPyP was not efficient for hydrogen peroxide clearance. Therefore, by reducing cytochrome c, Mn(II)TMPyP can indirectly contribute to hydrogen peroxide elimination.
Subject(s)
Antioxidants/chemistry , Cytochromes c/chemistry , Mitochondria, Liver/enzymology , Mitochondrial Membranes/enzymology , Porphyrins/chemistry , Animals , Rats , Rats, WistarABSTRACT
Oxidation of branched-chain amino acids (BCAAs) is tightly regulated in mammals. We review here the distribution and regulation of whole-body BCAA oxidation. Phosphorylation and dephosphorylation of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase complex directly regulates BCAA oxidation, and various other indirect mechanisms of regulation also exist. Most tissues throughout the body are capable of BCAA oxidation, and the flux of oxidative BCAA disposal in each tissue is influenced by three key factors: 1. tissue-specific preference for BCAA oxidation relative to other fuels, 2. the overall oxidative activity of mitochondria within a tissue, and 3. total tissue mass. Perturbations in BCAA oxidation have been implicated in many disease contexts, underscoring the importance of BCAA homeostasis in overall health.
Subject(s)
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/metabolism , Amino Acids, Branched-Chain/metabolism , Animals , Bacterial Proteins/metabolism , Decarboxylation , Female , Forecasting , Heart Failure/metabolism , Humans , Insulin Resistance/physiology , Male , Maple Syrup Urine Disease/genetics , Maple Syrup Urine Disease/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/enzymology , Multienzyme Complexes , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Organ Specificity , Oxidation-Reduction , Phosphorylation , Plant Proteins/metabolism , Protein Processing, Post-TranslationalABSTRACT
The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.
Subject(s)
Intestinal Mucosa/physiology , Mitochondria/physiology , Mitochondrial Membranes/enzymology , Mitochondrial Permeability Transition Pore/metabolism , Mytilus/enzymology , Proton-Translocating ATPases/physiology , Animals , Calcium/pharmacology , Cations/chemistry , Kinetics , Magnesium/pharmacology , Mitochondria/drug effects , Proton-Translocating ATPases/drug effects , Sulfides/pharmacologyABSTRACT
Assessment of activities of mitochondrial electron transport enzymes is important for understanding mechanisms of metabolic diseases, but structural organization of mitochondria and low sample availability pose distinctive challenges for in situ functional studies. We report the development of a tandem microfluidic respirometer that simultaneously tracks both the reduction of mediators on the electrode and the ensuing reduction of O2 by complex IV in the inner mitochondrial membrane. The response time of O2 consumption to multiple alternating potential steps is of approximately 10 s for a 150 µm-thick sample. Steady O2 depletion shows good quantitative correlation with the supplied electric charge, Pearson's r = 0.994. Reduction of mediators on biocompatible gold electrodes modified with carbon ink or fumed silica can compete with the oxidation of mediators by mitochondria, yielding an overall respiratory activity comparable to that upon chemical reduction by ascorbate. The dependence of O2 consumption on mediator and mitochondrial suspension concentrations shows that mass transport between the electrode and mitochondria does not limit biological activity of the latter. The mediated electrochemical approach is validated by the radiometric measurements of simulated changes in the intrinsic mitochondrial activity upon partial inhibition of complex IV by NaN3. This approach enables the development of O2-independent, biomimetic electrochemical assays narrowly targeting components of the electron transport chains in their native environments.
Subject(s)
Electrochemical Techniques , Electron Transport Complex IV/metabolism , Microfluidic Analytical Techniques , Mitochondria, Heart/enzymology , Mitochondrial Membranes/enzymology , Animals , Electron Transport , Oxygen/metabolism , RabbitsABSTRACT
Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.
Subject(s)
Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Sulfur/metabolism , Catalysis , Catalytic Domain , Cryoelectron Microscopy , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Hydrogenase/chemistry , Hydrogenase/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/metabolism , Models, Molecular , Origin of Life , Oxidation-Reduction , Proton Pumps/chemistry , Pyrococcus furiosus/chemistry , Pyrococcus furiosus/enzymology , Sodium-Hydrogen Exchangers/chemistryABSTRACT
Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.
Subject(s)
Mitochondrial Proton-Translocating ATPases/chemistry , Cryoelectron Microscopy , Membrane Lipids/chemistry , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/ultrastructure , Mitochondrial Proton-Translocating ATPases/classification , Mitochondrial Proton-Translocating ATPases/ultrastructure , Models, Molecular , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/chemistry , Proteins/chemistry , Proteins/ultrastructure , Protozoan Proteins/chemistry , Protozoan Proteins/ultrastructure , Tetrahymena thermophila/enzymology , Tetrahymena thermophila/ultrastructure , ATPase Inhibitory ProteinABSTRACT
Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Endoplasmic Reticulum/enzymology , Mitochondrial Membranes/enzymology , P-type ATPases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Cryoelectron Microscopy , HeLa Cells , Humans , P-type ATPases/genetics , Protein Conformation, alpha-Helical , Protein Domains , Saccharomyces cerevisiae/enzymology , Sequence AlignmentABSTRACT
The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer-monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer-monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.
Subject(s)
Mitochondria/enzymology , Mitochondrial Proton-Translocating ATPases , Animals , Cattle , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolism , Mitochondrial Proton-Translocating ATPases/ultrastructure , Models, Molecular , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Protons , TorqueABSTRACT
In the present study, we searched selective cytotoxicity and mitochondria mediated apoptosis of novel COX-2 inhibitor 2-(4-(Methylsulfonyl)phenyl)imidazo[1,2-a] pyridine-8-carboxylic acid on B-lymphocytes and their mitochondria isolated from normal subjects and acute lymphoblastic leukemia (ALL) patients' blood. Our results showed this compound can selectively induce cellular and mitochondrial toxicity on ALL B-lymphocytes and mitochondria without any toxic effects on normal B-lymphocytes and their mitochondria. Taken together, the results of this study suggest that cancerous mitochondria are a potential target for the ALL B-lymphocytes. Selective toxicity of COX-2 inhibitor in cancerous mitochondria could be an attractive therapeutic option for the effective clinical management of therapy-resistant ALL.
Subject(s)
B-Lymphocytes/drug effects , Cyclooxygenase 2 Inhibitors/pharmacology , Mitochondria/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , B-Lymphocytes/pathology , Caspase 3/metabolism , Cell Survival/drug effects , Child , Child, Preschool , Cytochromes c/metabolism , Female , Humans , Male , Membrane Potential, Mitochondrial/drug effects , Mitochondria/enzymology , Mitochondria/pathology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Reactive Oxygen Species/metabolism , Succinate Dehydrogenase/metabolismABSTRACT
Cardiotoxin CTII from Najaoxiana cobra venom translocates to the intermembrane space (IMS) of mitochondria to disrupt the structure and function of the inner mitochondrial membrane. At low concentrations, CTII facilitates ATP-synthase activity, presumably via the formation of non-bilayer, immobilized phospholipids that are critical in modulating ATP-synthase activity. In this study, we investigated the effects of another cardiotoxin CTI from Najaoxiana cobra venom on the structure of mitochondrial membranes and on mitochondrial-derived ATP synthesis. By employing robust biophysical methods including 31P-NMR and 1H-NMR spectroscopy, we analyzed the effects of CTI and CTII on phospholipid packing and dynamics in model phosphatidylcholine (PC) membranes enriched with 2.5 and 5.0 mol% of cardiolipin (CL), a phospholipid composition that mimics that in the outer mitochondrial membrane (OMM). These experiments revealed that CTII converted a higher percentage of bilayer phospholipids to a non-bilayer and immobilized state and both cardiotoxins utilized CL and PC molecules to form non-bilayer structures. Furthermore, in order to gain further understanding on how cardiotoxins bind to mitochondrial membranes, we employed molecular dynamics (MD) and molecular docking simulations to investigate the molecular mechanisms by which CTII and CTI interactively bind with an in silico phospholipid membrane that models the composition similar to the OMM. In brief, MD studies suggest that CTII utilized the N-terminal region to embed the phospholipid bilayer more avidly in a horizontal orientation with respect to the lipid bilayer and thereby penetrate at a faster rate compared with CTI. Molecular dynamics along with the Autodock studies identified critical amino acid residues on the molecular surfaces of CTII and CTI that facilitated the long-range and short-range interactions of cardiotoxins with CL and PC. Based on our compiled data and our published findings, we provide a conceptual model that explains a molecular mechanism by which snake venom cardiotoxins, including CTI and CTII, interact with mitochondrial membranes to alter the mitochondrial membrane structure to either upregulate ATP-synthase activity or disrupt mitochondrial function.
Subject(s)
Cobra Cardiotoxin Proteins/metabolism , Elapid Venoms/toxicity , Mitochondria, Heart/drug effects , Mitochondrial Membranes/drug effects , Mitochondrial Proton-Translocating ATPases/metabolism , Naja naja , Phospholipids/metabolism , Animals , Binding Sites , Cattle , Cobra Cardiotoxin Proteins/toxicity , Elapid Venoms/metabolism , Membranes, Artificial , Mitochondria, Heart/enzymology , Mitochondrial Membranes/enzymology , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Time FactorsABSTRACT
Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic FO domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The FO complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes. The FO membrane domain, essential in the enzyme machinery, also participates in the bioenergetic cost of synthesizing ATP and in the formation of the cristae, thus contributing to mitochondrial morphology. The recent enzyme involvement in a high-conductance channel, which forms in the inner mitochondrial membrane and promotes the mitochondrial permeability transition, highlights a new F-type ATP synthase role. Point mutations which cause amino acid substitutions in FO subunits produce mitochondrial dysfunctions and lead to severe pathologies. The FO variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.
Subject(s)
Adenosine Triphosphate , Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Mitochondrial Proton-Translocating ATPases , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , Humans , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolismABSTRACT
Mitochondrial respiratory chain complexes I, III, and IV can associate into larger structures termed supercomplexes or respirasomes, thereby generating structural interdependences among the individual complexes yet to be understood. In patients, nonsense mutations in complex IV subunit genes cause severe encephalomyopathies randomly associated with pleiotropic complex I defects. Using complexome profiling and biochemical analyses, we have explored the structural rearrangements of the respiratory chain in human cell lines depleted of the catalytic complex IV subunit COX1 or COX2. In the absence of a functional complex IV holoenzyme, several supercomplex I+III2 species coexist, which differ in their content of COX subunits and COX7A2L/HIGD2A assembly factors. The incorporation of an atypical COX1-HIGD2A submodule attenuates supercomplex I+III2 turnover rate, indicating an unexpected molecular adaptation for supercomplexes stabilization that relies on the presence of COX1 independently of holo-complex IV formation. Our data set the basis for complex I structural dependence on complex IV, revealing the co-existence of alternative pathways for the biogenesis of "supercomplex-associated" versus individual complex IV, which could determine physiological adaptations under different stress and disease scenarios.
Subject(s)
Electron Transport Complex IV/metabolism , Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Cell Line , HumansABSTRACT
Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.
Subject(s)
Evolution, Molecular , Mitochondrial Proteins/chemistry , Peptide Hydrolases/chemistry , Protozoan Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Animals , Arabidopsis/classification , Arabidopsis/enzymology , Arabidopsis/genetics , Conserved Sequence , Euglena gracilis/classification , Euglena gracilis/enzymology , Euglena gracilis/genetics , Euglena longa/classification , Euglena longa/enzymology , Euglena longa/genetics , Gene Expression , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Leishmania major/classification , Leishmania major/enzymology , Leishmania major/genetics , Mice , Mitochondria/enzymology , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Phylogeny , Protein Domains , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Trypanosoma brucei brucei/classification , Trypanosoma brucei brucei/geneticsABSTRACT
The mitochondrial respiratory chain supercomplex (mitoSC) is a complex super-assembly formed by free complexes on the mitochondrial inner membrane respiratory chain through the interaction between their subunits, mainly including mitoSCI1+III2+IV1-4, mitoSCI1+III2, mitoSCIII2+IV1-2, high molecular weight mitoSC (HMW mitoSC) and mitochondrial metacomplex (mitoMC). mitoSC has been shown to improve the efficiency of electron transport in the respiratory chain and reduce the production of reactive oxygen species. The species and content of mitoSC change in different tissues in aging and many mitochondria-related diseases. By summarizing the structure and function of mitoSC in different tissues of human and mammals, and the changes of mitoSC under conditions of aging, heart disease, type 2 diabetes, cancer and genetic defects, this review focuses on the effects of exercise on mitoSC and its related regulation mechanisms in order to offer an insight for exercise interventions in mitochondria-related diseases.
Subject(s)
Electron Transport , Exercise , Mitochondrial Membranes/enzymology , Animals , Humans , Mitochondria , Mitochondrial DiseasesABSTRACT
Trypanosomatids are unicellular parasitic protozoa. Many of the species of this genera cause severe diseases in human, such as Leishmaniasis, African trypanosomiasis and Chagas disease. These parasites possess a single reticular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast or kDNA. kDNA encodes few essential mitochondrial proteins but no tRNAs. Therefore, trypanosomatid mitochondrion import a full set of nucleus-encoded tRNAs for mitochondrial translation. Recent advances indicated that mitochondrial protein translocases, particularly the subunits of the ATOM complex, are involved in the import of a tRNA in Trypanosoma brucei. However, the global picture and the role of the translocase components of the mitochondrial inner membrane (TbTims) are not well understood. Here we investigated the relative abundance of 16 different tRNAs in the cytosolic and mitochondrial fractions isolated from the six TbTims knockdown cell lines. We found that knockdown of TbTim17, one of the primary components of the TbTIM complex, reduced the abundance of all of these tRNAs into mitochondria and increased their abundance in the cytosol. Depletion of TbTim62, a TbTim17 associated proteins, also reduced the relative abundance of most of these tRNAs into mitochondria except for tRNAleu, tRNAmet, and tRNAglu. Whereas, knockdown of other TbTims, like TbTim50 and two small TbTims, TbTim10 and TbTim8/13, didn't have any effect on tRNA abundance either in the cytosol or mitochondria. Depletion of any of these TbTims showed minimal effect on the levels of total tRNAs in T. brucei. Absolute quantification of tRNA levels revealed that TbTim17 knockdown reduced the levels of different tRNAs in mitochondria from 3-6% to 0.8-1.4%, which is equivalent to ~70% reduction in average. Whereas, TbTim62 depletion showed somewhat selective effect. Overall, our results suggest that TbTim17 and TbTim62 are essential for tRNA import that further makes a connection between the tRNA and protein import into mitochondria in T. brucei.